DNA synthesis rate changes during the S phase in mouse epidermis

The in vivo DNA synthesis rate throughout the S phase of mouse epidermal cells was investigated. Epidermal basal cells were isolated at various times of the day from normal animals injected with [3H]TdR 30 min before sacrifice, and from pulse-labelled animals with regenerating and growth-inhibited epidermis. The cells were analysed by DNA flow cytometry combined with cell sorting. Cells from successive fractions of the S phase were sorted on glass slides and subjected to quantitative [3H]TdR autoradiography. The results confirmed the presence of unlabelled (slowly replicating) cells in the S phase, the proportion of which was circadian stage-dependent with minimum values at midnight and in the early morning. The DNA synthesis rate throughout the S phase showed a general trend with high values in the mid-fractions, a pattern which was similar in normal and in growth perturbed epidermis. In the early morning the DNA synthesis rate pattern was bimodal with maxima both in the first and second half of the S phase, with a corresponding trough in mid-S. At this time of day the cell progression rate through S is at its maximum, indicating a relationship between the overall DNA synthesis rate and the rate distribution pattern through S.     

Effects of Pulse Labelling With Tritiated Thymidine On the Circadian Rhythmicity In Epidermal Cell-Cycle Distribution In Mice

The influence of pulse labelling with 50 °Ci tritiated thymidine ([³H]TdR) (2 μCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G₂ phases of the cell cycle were estimated from the obtained DNA frequency distributions. the proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [³H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [³H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G₂ and M phase during the first 32 hr after the injection. the number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 °Ci [³H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

Effect of Methotrexate On Cells In A Keratinized Epithelium With an Active Thymidine Salvage Pathway Assessed By Autoradiography*

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m², injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [³H]TdR and [³H]UdR were used as tracers for salvage and de nouo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. the autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. the blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. the decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [³H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.)     

Effects of pulse labelling with tritiated thymidine on the circadian rhythmicity in epidermal cell-cycle distribution in mice

The influence of pulse labelling with 50 microCi tritiated thymidine ( [3H]TdR) (2 microCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G2 phases of the cell cycle were estimated from the obtained DNA frequency distributions. The proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [3H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [3H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G2 and M phase during the first 32 hr after the injection. The number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 microCi [3H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

CIRCADIAN RHYTHMS IN MOUSE EPIDERMAL BASAL CELL PROLIFERATION

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G₂ phase DNA content were measured by flow cytometry of isolated basal cells, and the [³H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G₂ phase and traversed the G₂ phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G₂ phase and mitosis. Circadian variations in the duration of the S phase, the G₂ phase and mitosis were clearly demonstrated.     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING APPLICATION OF A SKIN IRRITANT (CANTHARIDIN)

The strong skin irritant cantharidin dissolved in benzene was applied to the back of hairless mice. Single cell suspensions of epidermal basal cells were obtained and flow microfluorometric measurements of cellular DNA content were made. Smears were made for autoradiography, and the [³H]TdR labelling index (LI) and mean grain count (MGC) were assessed up to 3 days after cantharidin application. Three successive peaks of cells with S phase DNA content accompanied by three LI peaks were observed. The first two peaks were follwed by peaks of cells in G₂ phase, indicating that after the acute cell injury caused by cantharidin the cells traversed the cell cycle in partial synchrony through two subsequent cell cycles, each of 10–12 hr duration. During this phase of rapid proliferation the LI reached the proportion of cells in S phase, contrary to what is observed in untreated mouse epidermis, where the labelled cells contribute to about half the proportion of cells with S phase DNA content. The first two peaks of cells in S phase and LI coincided with an increased MGC, whereas the third peak was accompanied by a MGC significantly below control values. This indicates that this latter peak is due to a longer DNA synthesis time rather than to a partially synchronized and increased cell proliferation. The duration of the G₁, S and G₂ phases seems to be reduced initially in rapidly proliferating epidermis.     

SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([³H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([³H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [³H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G₁ because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.     

S phase synchrony in monolayer CHO cultures

Parameters are described for reproducible S phase synchrony of Chinese hamster ovary cells growing in monolayer, adapting a method described by Tobey & Crissman [1] for CHO cells growing in suspension culture. Cells are collected at the G1/S boundary in hydroxyurea after reversal of an early G1 block induced by isoleucine deprivation. The entire population enters the S period within 60 min after removal of hydroxyurea and proceeds through the S period with minimal decay of synchrony, as evidenced by autoradiographic and rate studies on [³H]TdR uptake. In addition, a method is described for obtaining cells synchronized during two successive S periods. The presence of hydroxyurea during G1 does not measurably affect the rate of uptake of [³H]uridine or [³H]leucine into TCA-insoluble material; however, cultures released from the hydroxyurea block at 10 h incorporate slightly more [³H]uridine (but not [³H]leucine) in the next 6 h than cultures maintained in hydroxyurea over this interval. Delaying entry into S with hydroxyurea for as long as 15 h does not significantly change the initial rate or duration of DNA synthesis upon removal of hydroxyurea, arguing against the build-up of substances responsible for initiation of replicons. Furthermore, if DNA synthesis is delayed with hydroxyurea in one cell cycle, a constant minimal interval of 15 h elapses before the population enters into the next S phase, suggesting that the timing of the S period is coupled to the timing of the previous S.     

Modulation of serum-stimulated DNA synthesis in cultured human fibroblasts by cAMP

Density-dependent inhibition of growth of cultured human fibroblasts was associated with a 3- to 4-fold rise in the intracellular concentration of cyclic AMP (cAMP). Serum lowered cAMP levels in 2–5 min, with the low levels persisting for several hours. When quiescent fibroblast cultures were treated with 10% serum, the incorporation of [³H]TdR into DNA increased after a 10–16 h lag, reaching a peak by 20–24 h. Dibutyryl cyclic AMP (db-cAMP), when present throughout serum treatment, produced a dose-dependent inhibition of [³H]TdR incorporation. Half-maximal inhibition was seen with 0.1 mM db-cAMP. When db-cAMP or another cyclic nucleotide phosphodiesterase inhibitor, l-methyl-3-isobutylxanthine (SC-2964), was added together with serum to maintain elevated cAMP levels and after 4 h was replaced with fresh serum-containing medium, the wave of DNA synthesis induced by serum was not delayed. This implied that stimulation by serum could occur without an initial decrease in cAMP concentration. In contrast, db-cAMP added 8 h later than serum and not removed, inhibited [³H]TdR incorporation at the peak to the same extent as db-cAMP added together with serum. The inhibition decreased progressively when db-cAMP was added more than 8 h after serum. These results suggested that a cAMP-sensitive step occurred approx. 8 h after the addition of serum in mid-G1 of the cell cycle. Results obtained using fibroblasts synchronized at the G1/S boundary with hydroxyurea or exposed to db-cAMP for 24 h suggested that db-cAMP also inhibited TdR incorporation at the G1/S interphase or during S phase. Thus, whereas reduced cAMP concentrations did not appear to serve as an initial trigger for serum-stimulated DNA synthesis in human fibroblasts, db-cAMP and SC-2964, presumably by elevating cAMP levels, appeared to act in mid-G1 and possibly at the G1/S boundary or within S phase to inhibit thymidine incorporation.     

CELL POPULATION KINETIC PROFILE OF THE MOUSE THYMUS, AND THE CHANGES INDUCED BY PREDNISOLONE

The cell population kinetic parameters of the thymus in BALB/c mice have been estimated using stathmokinetic and [³H]TdR techniques in both control animals and animals treated with prednisolone. FLM data were analysed by computer using the Gilbert program. The study showed that prednisolone had an inhibitory effect mainly in the DNA synthesis phase and in G₁. Stathmokinetic data also showed a decrease in the cell birth rate and an increase in the apparent cell cycle time (or potential doubling time) after treatment. The labelling index, the mitotic index and the growth fraction were also decreased. The study also shows a good agreement between the data obtained by stathmokinetic and [³H]TdR techniques.     

Cell population kinetics in pig epidermis: further studies

The cell population kinetics of the epidermis were studied in 4-month-old pigs. Mitotic figures were confined to the basal cell (L1) and the first suprabasal cell layer (L2). The mitotic index (MI) was 0.17 +/- 0.04% for L1 and 0.08 +/- 0.03% for L2. Labelled nuclei were distributed throughout the viable epidermis, the majority (79.1 +/- 1.1%) were in L1 with 19.5 +/- 1.2% in L2. The labelling indices (LI) in layers L1 and L2 were 7.1 +/- 0.4% and 3.4 +/- 0.1%, respectively. After labelling with two injections of tritiated thymidine [3H]TdR separated by 90 min, the LI increased to 8.2 +/- 0.3% in L1 and to 4.0 +/- 0.2% in L2. This increased labelling confirmed that cell proliferation occurs in both layers, L1 and L2, of the epidermis. The cell production rate (K) in L1 and L2 had an upper limit of 10.7 +/- 1.0 and 6.2 +/- 1.8 cells per 1000 cells per hour respectively. The cell flow rate per hour (cell flux), into and out of the DNA synthesis phase (S), and the duration of DNA synthesis were determined from double-labelling studies with [3H]TdR and [14C]TdR. The cell flux into and out of S was identical and was calculated as 0.6 +/- 0.1%/hr (L1) and 0.5 +/- 0.1%/hr (L2). Values for tS varied from 8 to 10 hr. The cell turnover times (tT) were in the range 89-129 hr and 180-261 hr for L1 and L2, respectively. Log normal curves were fitted to the fraction labelled mitoses data for L1 and L2. Values for tS for cells in L1 and L2 were 9.8 hr and 11.9 hr, respectively. tG2 + 1/2tM was 7.2 hr in L1 and 9.1 hr in L2.     

Cell position dependence of labelling thymidine nucleotides using the de novo and salvage pathways in the crypt of small intestine

About twice as much tritiated thymidine ([3H]TdR) is taken up by cells at the bottom of the crypt of the small intestine as by the rapidly cycling mid-crypt cells. However, the uptake of tritiated deoxyuridine ([3H]UdR) is even throughout the crypt. Exogenous thymidine is incorporated about four times and eight times more efficiently than deoxyuridine by the cells in the mid-crypt and cells at the bottom of the crypt, respectively. However all S phase cells in the crypt appear to be capable of using either precursors, i.e. either the de novo or salvage pathway. Since methotrexate (1 or 5 mg/kg) inhibits (at 5 mg/kg completely) the uptake of [3H]UdR, but has no effect on [3H]TdR uptake, the de novo and salvage pathways appear to be independent. Within the precision of the methods used in the experiments the 3 hr inhibition of the de novo pathway of deoxythymidylic acid (dTMP) synthesis by methotrexate does not produce any increase in utilization of the salvage pathway measured by incorporation of [3H]TdR into DNA. The increased efficiency of thymidine utilization by crypt base cells is not attributable to differences in accessibility of thymidine; differences in the rate of DNA synthesis or the size of the nuclei. It appears that crypt base cells (which include the putative stem cells) are efficient scavengers of [3H]TdR, and this might be related to the level of thymidine kinase activity within the cells, and/or to changes in the availability of endogenous thymidine (break-down products) which compete with exogenous [3H]TdR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subclassification of cells in S phase in a partially synchronized cell system

Abstract. A circadian dependent delay in the incorporation of [³H]TdR into DNA, presumably due to variations in the intracellular pool of [³H]TdR derivatives, was found. It seems reasonable to relate this effect to a circadianally varying age distribution of cells in S phase.
At any given time the S phase cells showed large variations in DNA synthesis rate, but it was still possible to identify a mean diurnal variation in the DNA synthesis rate.
Differences in the ability of S phase cells to incorporate [³H]TdR are also discussed in relation to flow cytometrical measurements, and this contributes to the understanding of the commonly observed phenomenon that flow cytometry estimates of S-fractions are higher than those obtained with autoradiography.     

Effect of methotrexate on cells in a keratinized epithelium with an active thymidine salvage pathway assessed by autoradiography

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m2, injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [3H]TdR and [3H]UdR were used as tracers for salvage and de novo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. The autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. The blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. The decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [3H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.     

INACCURACY OF ESTIMATIONS OF S PHASE FRACTION BY REDUCTION IN CLONING EFFICIENCY WITH HYDROXYUREA OR TRITIATED THYMIDINE

The current hypothesis, that the fractional reduction of cloning efficiency in semi-solid culture systems induced by pretreatment of the cells with hydroxyurea (HU) or [³H]TdR equals the fraction of cells initially in S phase, is tested. A lymphoblastoid cell line, SK-L7, with known cell cycle kinetics was exposed to cytotoxic concentrations of HU or suicidal doses of [³H]TdR and then initiated in semi-solid and liquid culture. Although approximately 0.6 of the initial population was in S, 1-hr exposures of HU at concentrations of up to 10^-2 M failed to reduce subsequent cloning efficiency. the 1-hr exposure to HU did not reduce either the immediate cell number or the gross population doubling rate over 24 hr. A 24-hr exposure to 10^-3 M HU reduced the cloning efficiency by approximately 98%, confirming the drug's cytotoxic capability. [³H]TdR at doses of 100 μCi/ml for 20–40 min reduced the cloning efficiency by approximately 60 and 70%, respectively. Although no cytotoxicity immediately after exposure was observed in either case, gross population doubling rate in liquid culture was reduced. While HU failed to reduce subsequent cloning efficiency, [³H]TdR reduced cloning efficiency by approximately the fraction of initial cells in S. the above hypothesis, therefore, cannot be applied naïvely as a technique for quantitating the fraction of a clonogenic cell population in S phase.     

CELL PROLIFERATION KINETICS OF EPIDERMIS AND SEBACEOUS GLANDS IN RELATION TO CHALONE ACTION

Median S-phase lengths of pinna epidermis and sebaceous glands, and of epithelia from the oesophagus and under surface of the tongue of Albino Swiss S mice were estimated by the percentage labelled mitoses method (PLM). The 18.4 and 18.8 hr for the median length of S-phase for pinna epidermis and sebaceous glands respectively made it possible for these two tissues to be used experimentally for testing tissue specificity in chalone assay experiments. The 10.0 and 11.5 hr for oesophagus and tongue epithelium respectively made experimental design for chalone assay difficult when pinna epidermis was the target tissue. The results of the Labelling Index measured each hour throughout a 24-hr period showed no distinct single peaked diurnal rhythm for pinna epidermis and sebaceous glands. Instead a circadian rhythm with several small peaks occurred which would be expected if an S-phase of approximately 18 hr was imposed on the diurnal rhythm. This indicates that there may be very little change in the rate of DNA synthesis. The results are given for the assay in vivo of purified epidermal G₁ and G₂ chalones, and the 72–81% ethanol precipitate of pig skin from which they could be isolated. These experiments were performed over a time period which took into account the diurnal rhythm of activity of the mice as well as the S-phase lengths. Extrapolating the results with time of action of the chalone shows that the G₁ chalone acts at the point of entry into DNA synthesis and that the S-phase length was approximately 17 hr for both the pinna epidermis and sebaceous glands. This may be a more correct value since the PLM method overestimates the median S-phase length as it is known that in pinna skin the [³H]TdR is available to the tissues for 2 hr and true flash labelling does not take place. The previous reports that epidermal G₁ chalone acts some hours prior to entry into S-phase resulted from experiments on back skin where the S-phase is shorter and there is a pronounceddiurnal rhythm which could mask the chalone effect. The epidermal G, chalone had no effect on DNA synthesis even at different times in the circadian rhythm. Thus the circadian rhythms and S-phase lengths of the test tissues need to be considered when experiments are performed with chalones. Ideally, the target tissues selected for cell line specificity tests should have the same cell kinetics for the easier and more accurate assessment and interpretation of results. When the tissues have markedly different cell kinetics, experimental procedures and results need to be evaluated accordingly. The point of action of G, chalone can only be assessed if the effect is measured over the peak of incorporation of 13H]TdR into DNA. The results of the effects of skin extracts are analysed in relation to changes in the availability of i3H]TdR for the incorporation into DNA and to the possibility of there being two distinct populations of proliferating cells.     

Kinetic Differences Between Fed and Starved Chinese Hamster Ovary Cells

When Chinese hamster (CHO-K1) cells are grown as monolayer cultures, they eventually reach a population-density plateau after which no net increase in cell numbers occurs. the kinetics of aged cells in nutritionally deprived (starved) or density-inhibited (fed) late plateau-phase cultures were studied by four methods: (i) Reproductive integrity and cell viability were monitored daily by clonogenic-cell assay and erythrosin-b dye-exclusion techniques. (ii) Mitotic frequencies of cells from 18 day old cultures were determined during regrowth by analysing time-lapse video microscope records of dividing cells. (iii) Tritiated-thymidine ([³H]TdR) auto-radiography was used to determine the fractions of DNA-synthesizing cells in cultures entering plateau phase and during regrowth after harvest. (iv) the rate of labelled nucleoside uptake and incorporation into DNA was measured using liquid scintillation or sodium iodide crystal counters after labelling with [³H]TdR or [¹²⁵]UdR. Non-cycling cells in starved cultures accumulate primarily as G₁, phase cells. Most cells not in G₁ phase had stopped in G₂, phase. Very few cells (< 2%) were found in S phase. In contrast, about half of the cells in periodically fed cultures were found to be in DNA-synthetic phase, and the percentage of these S phase cells fluctuated in a manner reflecting the frequency of medium replacement. Populations of both types of plateau-phase cultures demonstrate extremely coherent cyclic patterns of DNA synthesis upon harvest and reculturing. They retain this high degree of synchrony for more than three generations after the resumption of growth. From these data it is concluded that nutritionally deprived (starved) late plateau-phase cells generally stop in either G₁, or G₂, phase, whereas periodically fed late plateau-phase cultures contain a very large fraction of cycling cells. Populations of cells from these two types of non-expanding cultures are kinetically dissimilar, and should not be expected to respond to extracellular stimuli in the same manner.     

A Method For Measuring the Generation Time and Length of Dna Synthesizing Phase of Clonogenic Cells In A Heterogenous Population

Information on the cell cycle of progenitor cells in haemopoietic tissue is useful for understanding population control under physiological and abnormal conditions. Unfortunately, methods that have been developed for measuring cell cycle parameters are applicable only to cells of homogenous populations and not to morphologically non-recognizable progenitor cells such as colony forming units (CFU) that are present at low frequency in a heterogenous population. to circumvent this difficulty, a method was developed to measure CFU cell cycle parameters based on specific killing of cells in S phase by [³H]thymidine ([³H]TdR). This was done by estimating the number of CFU killed following exposure of the cell suspension to [³H]TdR for various time periods. Since cycling CFU are continuously entering S phase, a linear curve relating the percentage CFU-kill to the length of exposure of the cells to [³H]TdR in culture can be obtained. the slope of the curve (percentage kill/hr) indicates the rate that CFU enter the S phase and travel through the cell cycle. the inverse of this value will then represent a time period for CFU to move through a complete cell cycle (generation time). the length of S phase can then be obtained by multiplying generation time by the fraction of cells in S phase at time zero. This method has been used to measure generation time and length of S phase of three kinds of haemopoietic progenitor cells: mouse granulocyte-macrophage CFU, human T lymphocyte CFU and CFU from regenerating mouse spleens. This method should be applicable to any normal or neoplastic clonogenic cell populations and the latter could be either of haematological or of solid tumour origin.     

Expression of the mitochondrial genome in HeLa cells. XXI. Mitochondrial protein synthesis during the cell cycle

Chloramphenicol sensitive [³H]leucine incorporation into protein (due to mitochondrial protein synthesis) in synchronized HeLa cells has been found to continue throughout interphase, its rate per cell approximately doubling from the G₁ to the G₂ phase. This increase in the rate of [³H]leucine incorporation during the cycle does not seem to parallel closely the increase in cell mass. In fact, the observations made on cultures incubated at 34.5 °C, where the G₁ and S phases are better resolved than at 37 °C, indicate that the rate remains constant during the G₁ phase, and starts to accelerate with the onset of nuclear DNA synthesis. Correspondingly, on a per unit mass basis, there appears to be a slight decline in the rate of [³H]leucine incorporation into protein during the G₁ phase, which is compensated by an increase in the early S phase. No significant variations were observed in the mitochondrial leucine pool labeling during the cell cycle; therefore, the observed pattern of [³H]leucine incorporation into protein should reflect fairly accurately the behavior of mitochondrial protein synthesis. Evidence has been obtained indicating a depression in the rate of incorporation of [³H]leucine into protein in mitochondria of mitotic cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the products of mitochondrial protein synthesis has not revealed any differences in the size distribution of the proteins synthesized in the various portions of the cell cycle.     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Cellular uptake of [3H]thymidine [( 3H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 X 10(-18) mol/cell. The pool activity of G1 cells was unmeasurable but rose to maximum values at the border of the G1-S phase. It decreased again during G2. The [3H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10% X min-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.